人类3-磷酸甘油醛脱氢酶的原核表达、纯化和鉴定

Prokaryotic Expression,Purification and Identification of Human Glyceraldehyde 3-phosphate Dehydrogenase

为了研究3-磷酸甘油醛脱氧酶(GAPDH)化生物学功能,以pcDNA3.1-GAPDH质粒为模板,PCR方法扩增GAPDH基因,经BamHI和SalI双酶切后插入相同酶切的pET32a(+)载体,构建His-GAPDH融合蛋白表达质粒pET32a(+)-GAPDH;转化JM109感受态细胞,并进行阳性克隆筛选,扩增目的质粒;转化大肠杆菌BL21菌株,经IPTG诱导产生融合蛋白,亲和层析柱纯化后,用SDS-PAGE和Western blotting检测GAPDH蛋白表达情况。结果表明,构建的人GAPDH基因表达载体经序列测定证实,与GenBank数据完全一致;双酶切鉴定证实,克隆基因正确插入pET32a(+)载体;表达质粒pET32a(+)-GAPDH在大肠杆菌BL21中成功地诱导表达了可溶性His-GAPDH融合蛋白,纯化后SDS-PAGE和Western blotting检测证实融合蛋白表达成功。人GAPDH原核表达载体的成功构建,及6His-GAPDH融合蛋白的正确表达,为进一步深入研究GAPDH的生物学功能奠定了基础。

The prokaryotic expression vector pET32a （＋）-GAPDH and the expression of human glyceraldehyde 3- phosphate dehydrogenase （GAPDH）in E.coli were investigated. GAPDH gene was arnphfied by PCIK from pcDNA3.1- GAPDH plasmid and inserted into the plasmid pET32a（＋）followed by digestion with BamHI and SalI. The recombinant plasmid pET32a （＋）-GAPDH was transformed into E.coli JM109 and selected with ampicillin. The positive clones containing the recombinant plasmid pET32a （＋）-GAPDH were verified. His-GAPDH fusion protein was expressed in E. coli BL21 （ED3） by IPTG induction,and was identified by SDS-PAGE and Western Noting. Results showed that the PCR product of human glyceraldehyde 3-phosphate dehydrogenase （GAPDH） was identical to the sequence in GenBank and was accurately inserted into the pET32a （＋）. SDS-PAGE and Western blotting indicated that the dissoluble fusion protein successfully expressed in E.coli BL21 （ED3）. Therefore,a prokaryotic vector for human GAPDH gene has been successfully constructed and 6His-GAPDH has been correctly expressed,the study of which may facilitate subsequent research of its biological functions.